PROJECT SUMMARY The primary cilium, an antenna-like structure on the surface of most quiescent and differentiated cells, is essential for transducing signals required for growth control, including Hedgehog (Hh) signals. Consequently, loss of cilia alters responsiveness to extracellular cues. Many cancers, including breast cancer, exhibit near- complete loss of cilia during the early stages of oncogenic transformation. Novel strategies are urgently needed to treat the most lethal sub-type, triple-negative breast cancer (TNBC). TNBC is disproportionately observed in African-Americans and thus represents a key health disparity. We propose that restoring ciliogenesis to breast cancer cells will restore growth suppressive signaling and block tumor growth. Our preliminary data indicate that (1) cancer cells can regrow cilia; (2) single gene or pharmacological intervention can cause cancer cells to regrow cilia; and (3) ciliary regrowth inhibits proliferation. We will build on these findings by conducting an innovative functional screen to identify the genes that restrict ciliogenesis in breast cancer cells, and test whether restoring ciliogenesis limits tumor growth in vivo. The identified ciliogenesis inhibitors will help unravel the molecular mechanisms by which cancer cells suppress ciliogenesis, identify how cilia inhibit breast cell proliferation, and test, for the first time, whether reversing the loss of cilium-associated signaling restores growth regulation to transformed breast cancer cells. In Aim 1, we will perform a novel genome-wide screen to identify inhibitors of ciliogenesis in breast cancer. Prior screens have identified regulators that promote ciliogenesis. In contrast, our screen will identify negative regulators of cilium assembly. We will also test the innovative hypothesis that restoration of cilia in combination with use of clinically relevant inhibitors of ciliary signaling can block tumor growth. In Aim 2, we will test how restoring ciliogenesis impacts breast cancer growth in animal models. Together, these studies will reveal how cancer cells restrict ciliogenesis, an important, unanswered question in cancer cell biology. The successful demonstration that restoring ciliogenesis inhibits tumor growth will reveal a novel strategy for targeting breast cancer, which may be applicable to other cancer types. By taking advantage of the complementary strengths of the Dynlacht and Reiter laboratories, we will innovatively combine RNAi screening, state-of-the-art ciliary analysis, and organismal cancer modeling. While highly exploratory, in keeping with the R21 mechanism, discovering the means to promote ciliogenesis and restrain breast cancer growth will identify novel ways that tumors avoid growth control and may establish a novel paradigm for treating breast cancer. Furthermore, to address cancer health disparities, we will: (1) begin to lay the mechanistic foundation for understanding why TNBC disproportionately affects African-Americans and (2) acquire and characterize primary TNBC samples to determine whether tumor ciliation or expression of regulators of ciliogenesis differs in different ethnic populations. !